IPV-DPT vaccine

ABSTRACT

The invention provides a process for producing a combined vaccine containing an inactivated Sabin strain of poliovirus, a  Bordetella pertussis  protective antigen, a diphtheria toxoid and a tetanus toxoid, the process including a step of producing a high-titer Sabin strain poliovirus. The inventive process for producing a combined vaccine, including a step of culturing, in the presence of from about 4 g/L to about 6 g/L of a microcarrier, Vero cells to be inoculated with a Sabin strain of poliovirus, is useful as a process for efficiently producing a combined vaccine containing an inactivated Sabin strain of poliovirus.

TECHNICAL FIELD

The present invention relates to a combined vaccine, particularly acombined vaccine containing an inactivated Sabin strain of poliovirus(sIPV), and to a method of preparation thereof

BACKGROUND ART

Polio is an infectious disease caused by a poliovirus. Poliovirusesinfect human by an oral route, proliferate in the intestinal tract, andenter the central nervous system through the blood. The proliferationwithin large motor neutrons of polioviruses that have entered thecentral nervous system causes neuronal degeneration and necrosis,triggering acute flaccid paralysis in the limbs. Moreover, when thepoliovirus affects the medullary respiratory center, death fromrespiratory paralysis may result. Polio vaccines are widely used tosuppress the onset of polio which triggers such severe symptoms.

Two types of polio vaccines are used: oral live polio vaccines andinactivated polio vaccines. Oral live polio vaccines are vaccines whichuse attenuated strains of poliovirus (Sabin strains). Attenuated strainsof poliovirus that administered orally cause normal infections.Polioviruses from oral live polio vaccines grow well in the intestines,resulting in the formation of localized immunity within the intestines.In addition, when polioviruses from an oral live polio vaccine enter theblood and cause viremia, this also stimulates the production ofantibodies within the blood. However, because the ability of attenuatedstrains of polioviruses to proliferate within the central nervous systemis very weak, they generally do not cause paralysis. In the body of theinoculee, polioviruses from the oral live polio vaccine multiply and areexcreted in the stools about 4 to 6 weeks following inoculation. Theexcreted viruses will infect people around the inoculee who have a weakimmunity or no immunity to polio, conferring immunity or exhibiting apotentiating effect in the same way as in the inoculee.

However, in the course of repeated growth within the body of theinoculee or in the course of repeated growth in the body of a personinfected by excreted viruses, an attenuated strain of poliovirus from anoral live polio vaccine sometimes gives rise to mutations in a highlyvirulent direction. In very rare instances, such mutants causevaccine-associated paralysis.

An inactivated polio vaccine is a vaccine which has lost itsinfectiousness by inactivation of the poliovirus with formalin. Becausean inactivated polio vaccine neither multiplies within the body of theinoculee nor infects people around the inoculee, it will not causevaccine-associated paralysis. Highly virulent strains have hitherto beenused to prepare inactivated polio vaccines, but advances have also beenmade recently in the development of attenuated strains (Sabin strains)(Biologicals 34, 151-154 (2006); Dev. Bil. Basel. Karger 105, 163-169(2001), Clinical Virology 30, No. 5, 336-343 (December 2002)). Thesomewhat poorer growth of attenuated strains (Sabin strains) than highlyvirulent strains has been regarded as a drawback.

Vaccines which contain a Bordetella pertussis protective antigen, adiphtheria toxoid and a tetanus toxoid are widely used asdiphtheria-tetanus-pertussis combined vaccines.

Polyvalent vaccines composed of an acellular pertussis vaccine, adiphtheria toxoid, a tetanus toxoid and inactivated poliovirus are known(Published Japanese Translation of a PCT Application No. 2000-504032).

Vero cells are passage cells from the kidneys of green monkeys. Becausethese cells have a broad sensitivity to various types of viruses, theyare widely used in virus cultivation. A method for preparing anenterovirus type 71 vaccine that been reported in the literatureincludes culturing Vero cells on a microcarrier and using the culturedVero cells to grow enterovirus 71 (“Optimization of microcarrier cellculture process for the inactivated enterovirus type 71 vaccinedevelopment,” by Suh-Chin Wu, et al.: Vaccine 22, 3858-3864 (2004)).General cell culturing conditions using a microcarrier have also beendescribed (Microcarrier cell culture principles & methods: PharmaciaLKB, Biotechnology, 1988).

DISCLOSURE OF THE INVENTION

Under such circumstances, there has existed a desire for a process forproducing a combined vaccine containing an inactivated Sabin strain ofpoliovirus (sIPV), and also containing a B. pertussis protectiveantigen, a diphtheria toxoid and a tetanus toxoid (DPT), which processincludes the step of preparing a high-titer Sabin strain poliovirus.

The inventors have conducted extensive investigations in order toresolve the above problem. As a result, the inventors have discoveredthat a high-titer Sabin strain poliovirus can be prepared by culturing,in the presence of from about 4 g/L to about 6 g/L of a microcarrier,Vero cells inoculated with a Sabin strain of poliovirus. Afterconducting repeated investigations based on these findings, theinventors ultimately arrived at the present invention.

The present invention thus provides such as:

-   (1) A process for preparing a combined vaccine containing

(A) an inactivated Sabin strain of poliovirus,

(B) a Bordetella pertussis protective antigen,

(C) a diphtheria toxoid, and

(D) a tetanus toxoid,

the process comprising (a) a step of culturing, in the presence of fromabout 4 g/L to about 6 g/L of a microcarrier, Vero cells to beinoculated with a Sabin strain of poliovirus.

-   (2) The process according to (1) above, further comprising:

(b) a step of infecting the Vero cells with a Sabin strain ofpoliovirus;

(c) a step of allowing the poliovirus to proliferate;

(d) a step of recovering a virus fluid containing the poliovirus; and

(e) a step of inactivating the poliovirus.

-   (3) The process according to (1) above, wherein the microcarrier has    a concentration of about 5 g/L.-   (4) The process according to (1) above, wherein the microcarrier is    a dextran microcarrier.-   (5) The process according to (1) above, wherein the step growing the    Vero cell (step (a)) is carried out on a scale of at least about 3    liters.-   (6) The process according to (1) above, wherein the step of growing    the Vero cell (step (a)) is carried out on a scale of at least about    30 liters.-   (7) The process according to (2) above, further comprising

(d-2) a step of purifying the virus fluid.

-   (8) The process according to (7) above, wherein the purifying step    (step (d-2)) comprises:

(i) forming the virus fluid recovered in step (d) into a pellet byultracentrifugation;

(ii) sonicating a re-suspension of the pellet; and

(iii) purifying by column chromatography.

-   (9) The process according to (8) above, wherein the purification by    column chromatography (iii) is carried out one time only.-   (10) A vaccine prepared by the process according to (1) above.-   (11) The process of (1) above, wherein the step of culturing the    Vero cell (step (a)) is carried out on a scale of at least about 3    liters.-   (12) The process of (1) above, wherein the step of culturing the    Vero cell (step (a)) is carried out on a scale of at least about 30    liters.

By culturing, in the presence of from about 4 g/L to about 6 g/L of amicrocarrier, the Vero cells to be inoculated with a Sabin strain ofpoliovirus, it is possible to obtain high-titer Sabin strain poliovirus.By using the high-titer Sabin strain poliovirus, an inactivated Sabinstrain poliovirus can be efficiently produced. Therefore, a process forproducing a combined vaccine which includes the step of culturing, inthe presence of from about 4 g/L to about 6 g/L of a microcarrier, Verocells to be inoculated with a Sabin strain of poliovirus (the productionprocess of the present invention) is useful as a process for theefficient production of combined vaccines containing an inactivatedSabin strain of poliovirus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing Vero cell growth curves in the culturing ofVero cells by a microcarrier method. Here, “3L” indicates a startingcell number of about 2×10⁵ cells/mL (low concentration) and 3 g/L ofmicrocarrier. “3H” indicates a starting cell number of about 10×10⁵cells/mL (high concentration) and 3 g/L of microcarrier. “5L” indicatesa starting cell number of about 2×10⁵ cells/mL (low concentration) and 5g/L of microcarrier. “5H” indicates a starting cell number of about10×10⁵ cells/mL (high concentration) and 5 g/L of microcarrier.

FIG. 2 is a graph showing the infectivity titers (virus titers) ofpoliovirus type I obtained in Vero cells cultured under variousconditions. Here, “3L” represents type I polioviruses obtained in Verocells cultured from a starting cell number of about 2×10⁵ cells/mL (lowconcentration) on 3 g/L of microcarrier. “5L” indicates type Ipolioviruses obtained in Vero cells cultured from a starting cell numberof about 2×10⁵ cells/mL (low concentration) on 5 g/L of microcarrier.“5H” indicates type I polioviruses obtained in Vero cells cultured froma starting cell number of about 10×10⁵ cells/mL (high concentration) on5 g/L of microcarrier. “Ref.” indicates type I polioviruses grown usinggreen monkey kidney cells.

The present invention provides a process for producing a combinedvaccine containing an inactivated Sabin strain of poliovirus (sIPV)together with a B. pertussis protective antigen, a diphtheria toxoid anda tetanus toxoid (DPT), which process includes the step of producing ahigh-titer Sabin strain of poliovirus.

The invention is described more fully below.

1. Inactivated Sabin Strain Poliovirus

(1) Sabin Strain Poliovirus

In the present specification, “Sabin strain of poliovirus” refers to apoliovirus strain derived from an attenuated strain of poliovirusisolated by Dr. Albert B. Sabin (see, for example, Sabin, A B, Boulger,L R: “History of Sabin attenuated poliovirus oral live vaccine strains,”J. Biol. Standard 1, 115-118 (1973)).

Sabin strain polioviruses include Sabin type I strains of poliovirus,Sabin type II strains of poliovirus and Sabin type III strains ofpoliovirus. Examples of Sabin type I strains of poliovirus include thestrains LSc and 2ab. Examples of Sabin type II strains of poliovirusinclude the strains P712, Ch and 2ab. Examples of Sabin type III strainsof poliovirus include the strains Leon and 12a₁b.

(2) Inactivation

In the present specification, virus “inactivation” refers to eliminatingthe infectious ability of a virus. Methods of inactivation include, butare not limited to, physical methods (e.g., methods involving the use ofx-ray irradiation, heat or ultrasound) and chemical methods (e.g.,methods involving the use of formalin, mercury, alcohol or chlorine).

Poliovirus inactivation may be carried out by a known method (see, forexample, Biologicals 34, 151-154 (2006)). For instance, inactivation maybe carried out by treating the poliovirus with formalin.

(3) Immunogenicity of Inactivated Sabin Strains of Poliovirus

Two viral antigens known as D antigens and C antigens are generallypresent in admixture within inactivated Sabin strains of poliovirus. Dantigens are complete viral particles. Antibodies to D antigens have theability to neutralize the infectiousness of live viruses, and functionas protective antibodies. C antigens, called defective particles, areparticles missing the core nucleic acid RNA and part of the viralproteins of a complete viral particle; these particles are hollow at thecenter. Antibodies to C antigens have little or no ability to neutralizethe infectiousness of live viruses. Therefore, when an inactivated Sabinstrain of poliovirus is to be used as a vaccine, the D antigens arerequired.

There are three types of polioviruses: type I, type II and type III. Theimmunity to poliovirus infection is specific to the three virustypes—type I, type II and type III; what cross-immunity may existbetween types is minimal. The D antigens of inactivated type I, type IIand type III Sabin strains of poliovirus have immunogenicities capableof producing neutralizing antibodies for, respectively, type I, type IIand type III wild strains (highly virulent) of poliovirus. Theimmunogenicities of the D antigens of inactivated Sabin strains ofpoliovirus differ for each of types I, II and III; hence, the amount ofD antigen required to produce enough antibody to neutralize type I, typeII and type III wild strains (highly virulent) of poliovirus differsaccording to the virus type.

As mentioned above, there are three types of polioviruses—type I, typeII and type III, each of which causes the same polio. Therefore, when aninactivated Sabin strain of poliovirus is used as a vaccine (inactivatedpolio vaccine), it must have an immunogenicity capable of producingsufficient antibodies to neutralize wild strains (highly virulentstrains) of polioviruses of each of types I, II and III. Moreover, it isdesirable for the immunogenicity to be close to the immunogenicity ofthe inactivated polio vaccines from highly virulent strains that havehitherto been used. To exhibit such an immunogenicity, the vaccinepreferably contains type I, type II and type III inactivated Sabinstrains of poliovirus in a proportion, by weight of the respective Dantigens, of preferably (2 to 4):(80 to 120):(80 to 120), and mostpreferably (about 3):(about 100):(about 100). The inactivated Sabinstrains of poliovirus used in the present invention, by including typesI, II and Ill in a specific proportion like the foregoing, exhibits animmunogenicity similar to that of inactivated polio vaccines from highlyvirulent strains (e.g., the Sauk vaccine).

2. Production of Inactivated Sabin Strains of Poliovirus

Inactivated Sabin strains of poliovirus suitable for use in the presentinvention can be produced by the method described below.

First, Vero cells are cultured in the presence of from about 4 g/L toabout 6 g/L of microcarrier, thereby giving cells for culturingpolioviruses. The resulting poliovirus culturing cells are inoculatedwith seed viruses (Sabin strain polioviruses) and the viruses arecultured, yielding Sabin strain polioviruses that have proliferated. Theresulting Sabin strain polioviruses are inactivated to give inactivatedSabin strain polioviruses. Inactivated Sabin strain polioviruses can beobtained which correspond to the seed viruses used (Sabin type Istrains, Sabin type II strains or Sabin type III strains). The virusesmay be concentrated and/or purified before or after virus inactivation.

As mentioned above, the inactivated polio vaccine must have animmunogenicity capable of producing sufficient antibodies to neutralizewild strains (highly virulent strains) of each of types I, II and III.However, the D antigens of inactivated poliovirus Sabin strains haveimmunogenicities that differ between types I, II and III. Accordingly,inactivated polio vaccine possessing an immunogenicity capable ofproducing sufficient antibodies to neutralize wild strains (highlyvirulent strains) of each of types I, II and III can be obtained byadjusting the amounts of type I, type II and type III inactivatedpolioviruses contained.

(Vero Cells)

Vero cells are passage cells from the kidneys of green monkeys(Cercopithecus aethiops), and are deposited with the American TypeCulture Collection (ATCC). Vero cells are widely used to culture virusesbecause they exhibit a fibroblastic morphology, have a broad sensitivityto various types of viruses and are easy to maintain as passage cells.Vero cells that are known to be available from ATCC include ATCC Nos.CCL-81 and CRL-1587.

(Microcarrier)

In this specification, “microcarrier” refers to a carrier that hassurfaces to which cells adhere and enables cell cultivation to becarried out in a suspended state within a liquid medium. Themicrocarrier is not subject to any particular limitation with regard tomaterial, shape and size, provided it is a carrier to the surface ofwhich cells adhere and which enables cell cultivation to be carried outin a suspended state within a liquid medium.

Examples of the microcarrier material include dextran, gelatin,collagen, polystyrene, polyethylene, polyacrylamide, glass andcellulose. Dextran is preferred as the microcarrier material.

Examples of the microcarrier shape include spherical (beads) anddiscoidal shapes. The microcarrier preferably has a spherical shape.

The spherical microcarrier has a size of, for example, from about 0.01to about 1 mm, preferably from about 0.05 to about 0.5 mm, and morepreferably from about 0.1 to about 0.3 mm.

The microcarrier may be porous.

Examples of dextran spherical microcarriers that may be used in thepresent invention include CYTODEX 1 (trade name), CYTODEX 3 (trade name)and CYTOPORE (trade name) (all products of GE Healthcare Biosciences).Examples of discoidal microcarriers include CYTOLINE 1 (trade name) andCYTOLINE 2 (trade name) (both products of GE Healthcare Biosciences).Examples of porous microcarriers include CYTOPORE (trade name), CYTOLINE1 (trade name) and CYTOLINE 2 (trade name) (all products of GEHealthcare Biosciences). The microcarrier used in the present inventionis most preferably a spherical dextran microcarrier. The sphericaldextran microcarrier is preferably CYTODEX 1 (trade name), CYTODEX 3(trade name) or CYTOPORE (trade name), more preferably CYTODEX 1 (tradename) or CYTODEX 3 (trade name), and most preferably CYTODEX 1 (tradename).

(Vero Cell Cultivation)

In the present invention, the Vero cells are grown by cultivation in thepresence of from about 4 g/L to about 6 g/L of microcarrier. Themicrocarrier concentration is preferably from about 4.5 g/L to about 5.5g/L, and more preferably about 5 g/L.

The above-described Vero cell growing step is carried out on a scale, interms of the liquid volume, of preferably at least 3 liters, morepreferably at least 30 liters, and most preferably at least 150 liters.The Vero cell growing step is carried out on a scale of generally notmore than 1,000 liters.

When the Vero cells are cultivated in the presence of a microcarrier,the culture medium used may be, for example, ME medium (Science, 122,501 (1952)), DME medium (Virology, 8, 396 (1959)), RPMI 1640 medium (TheJournal of the American Medical Association, 199, 519 (1967)) or 199medium (Proceedings of the Society for the Biological Medicine, 73, 1(1950)), which contain from about 5 to about 20 vol % of calf serum orfetal bovine serum. The culture medium is preferably a DME medium, morepreferably a DME medium containing calf serum, and most preferably a DMEmedium containing about 5 vol % of calf serum. The medium may, ifnecessary, be changed during the period of cell cultivation. The pH ispreferably from about 6 to about 8, more preferably from about 6.5 toabout 7.5, and most preferably about 7. Cultivation is typically carriedout at from about 35° C. to about 40° C. for a period of from about 5 to9 days. If necessary, aeration and stirring may be carried out duringcultivation. The dissolved oxygen concentration (DO) at the time of cellcultivation is preferably from about 60 to about 90%, more preferablyfrom about 70 to about 80%, and most preferably about 75%.

The number of Vero cells in the medium at the start of cultivation inthe presence of the microcarrier (starting number of cells) may be setas appropriate for such factors as the type of medium, the type ofmicrocarrier, the scale of cultivation. The starting number of cells ispreferably from 2×10⁴ cells/mL to 10×10⁵ cells/mL, and most preferablyfrom 2×10⁵ cells/mL to 10×10⁵ cells/mL.

(Poliovirus Cultivation)

Poliovirus cultivation may be carried out by inoculating and thusinfecting the cultured Vero cells with a Sabin strain of poliovirus(seed virus), and culturing the poliovirus within the cells. Cultivationof the infected cells may be carried out in the same way as theabove-described Vero cell cultivation. The medium used for polioviruscultivation is preferably a 199 medium, more preferably a 199 mediumcontaining sodium bicarbonate, and most preferably a 199 mediumcontaining 0.3 w/v % of sodium carbonate.

The incubation temperature during virus cultivation is preferably fromabout 30° C. to about 38° C., more preferably from about 32° C. to about36° C., and most preferably from about 33° C. to about 35° C. The viruscultivation period is preferably from about 1 to about 5 days, morepreferably from about 2 to about 4 days, and most preferably about 3days. Virus cultivation may be brought to an end using cytopathiceffects by the poliovirus (rounding of the poliovirus-infected cells anddetachment of the cells from the microcarrier) as the indicator.

A Sabin strain of poliovirus that has been cultivated using green monkeyprimary-cultured kidney cells may be employed as the seed virus.

(Recovery of Poliovirus-Containing Virus Fluid)

Following the completion of virus cultivation, the microcarrier isremoved and the virus fluid containing the poliovirus (sometimesreferred to herein as the “poliovirus fluid”) is recovered.

Removal of the microcarrier may be carried out by means of, for example,a Teflon mesh (such as one having a pore size of 120 μm). Becausepolioviruses remain present (attached) to the microcarrier left on themesh, these remaining polioviruses may be recovered by rinsing with, forexample, the virus culture medium.

The resulting poliovirus fluid may be filtered using a filter membrane(e.g., a 0.2 μm filter membrane) or the like to remove cell debris.

The poliovirus fluid may be concentrated and/or purified before or afterinactivation.

Illustrative examples of the method of concentration includeultrafiltration, ultracentrifugation and dialysis. The method ofconcentration is preferably ultrafiltration or ultracentrifugation. Itis more preferable to carry out both ultrafiltration andultracentrifugation, and even more preferable to carry outultrafiltration followed by ultracentrifugation.

The membrane used in ultrafiltration may be an ultrafiltration membranecommonly employed for concentrating viruses. The ultrafiltrationmembrane has a molecular weight cutoff which is preferably about 100kDa. The ultrafiltration membrane is preferably made of a material suchas polyether sulfone.

Concentration of the poliovirus by ultracentrifugation may be carriedout by subjecting the poliovirus fluid to 4 hours of centrifugation at4° C. and 100,000 g to form a pellet. The pellet may be re-suspended in,for example, a phosphate buffer. It is also possible to break up themass of aggregated viruses by sonicating the pellet re-suspension.Sonication may be carried out using a commercially available apparatus,such as Insonator Model 200M (Kubota). The sonication conditions shouldbe sufficient to break up the mass of aggregated viruses, and may beselected as appropriate for such factors as the vessel used insonication, the ultrasound output, and the concentration of there-suspension. Sonication conditions are exemplified by treatment at 200W for a period of from 3 to 10 minutes. Even when such sonication iscarried out, the Sabin strain of poliovirus does not lose itsimmunogenicity, enabling it to be advantageously used as a poliovirusvaccine.

Methods of purification include, but are not limited to, methods whichutilize physical characteristics of the substance being purified, suchas size, density and sedimentation coefficient, and methods whichutilize chemical or physicochemical reactions (e.g.,adsorption-desorption). Illustrative examples of methods of purificationinclude density-gradient centrifugation, filtration (includingultrafiltration), ion-exchange column chromatography, affinitychromatography, gel filtration chromatography and salting out. Themethod of purification is preferably column chromatography, morepreferably ion-exchange column chromatography, and most preferablyDEAE-ion-exchange column chromatography. The number of timespurification is carried out by column chromatography is not subject toany particular limitation. That is, purification by columnchromatography may be carried out repeatedly until the required purityis achieved. However, from the standpoint of production efficiency andother considerations, it is preferable to carry out such purification inas few steps as possible.

Concentration and purification are preferably carried out by forming thepoliovirus fluid into a pellet by ultracentrifugation, sonicating are-suspension of the resulting pellet, then purifying by columnchromatography. In addition, from the standpoint of productionefficiency, it is preferable to carry out purification by columnchromatography one time only. By carrying out formation of thepoliovirus fluid into a pellet by ultracentrifugation, sonication of are-suspension of the pellet and purification by column chromatographyone time only, the poliovirus fluid can be both efficiently andadequately concentrated and purified.

(Inactivation of Poliovirus)

Inactivation of the poliovirus may be carried out by a commonly employedmethod. Specifically, the poliovirus may be inactivated by adding aninactivator to the poliovirus fluid and thereby effecting a reactionbetween the poliovirus and the inactivator. The inactivator ispreferably formalin. The inactivation conditions are not subject to anyparticular limitation, so long as the poliovirus is inactivated. Toavoid the residual presence of insufficiently inactivated polioviruses,the period of inactivation treatment is generally from about 2 to about4 times, preferably from about 2.5 to about 3.5 times, and morepreferably about 3 times, the length of the period for which poliovirusinactivation has been confirmed.

For example, when formalin is used as the inactivator, the amount ofaddition is preferably from about 0.001 to about 0.1 w/v %, morepreferably from about 0.005 to about 0.05 w/v %, and most preferablyabout 0.01 w/v %. The inactivation temperature is most preferably about37° C. The inactivation period may vary also depending on the type ofinactivator, the concentration of the inactivator, and the inactivationtemperature. For example, when about 0.01 w/v % of formalin is used asthe inactivator and the inactivation temperature is about 37° C., theinactivation period is preferably from about 8 to about 16 days, morepreferably from about 10 to about 14 days, and most preferably about 12days. When about 0.01 w/v % of formalin is used and the inactivationtime is about 37° C., Sabin strain polioviruses are generallyinactivated within 4 days.

3. Diphtheria Toxoid, B. pertussis Protective Antigen, and TetanusToxoid (DPT)

The B. pertussis protective antigen, diphtheria toxoid and tetanustoxoid used in the present invention are not subject to any particularlimitation.

The B. pertussis protective antigen, diphtheria toxoid and tetanustoxoid are commercially available as diphtheria-pertussis-tetanuscombined vaccines (such as those manufactured by Takeda ChemicalIndustries, Ltd., the Research Foundation for Microbial Diseases ofOsaka University (Biken), and the Chemo-Sero-Therapeutic ResearchInstitute (Kaketsuken)). Alternatively, the B. pertussis protectiveantigen, diphtheria toxoid and tetanus toxoid can be obtained by knownmethods. Specifically, the B. pertussis protective antigen may beobtained by, for example, extracting, isolating and purifying thephylactic antigen fraction from a culture broth of strains of phase I B.pertussis (Tohama strain) using a physicochemical method such asammonium sulfate fractionation/sucrose density gradient centrifugalfractionation, then attenuating the remaining virulence with formalin.The diphtheria toxoid may be obtained by, for example, purifying andconcentrating the toxin produced by Corynebacterium diphtheriae(Park-Williams No. 8 strain) using a physicochemical method such ascolumn chromatography, followed by detoxification with formalin. Thetetanus toxoid may be obtained by, for example, purifying andconcentrating the toxin produced by Clostridium tetani (Harvard strain)using a physicochemical method such as column chromatography, followedby detoxification with formalin.

The B. pertussis protective antigen contains pertussis toxin (PTantigen), filamentous hemagglutinin (FHA antigen), outer membraneprotein (69 KD antigen), and fimbriae (also called FB antigen,agglutinogen (FGG)). The B. pertussis protective antigen need notnecessarily contain each of the above antigens, so long as it containsat least one, preferably at least two, and more preferably at leastthree of these antigens. Antibodies for these protective antigensprotect the host from pertussis.

4. Combined Vaccine

The combined vaccine of the present invention contains inactivated Sabinstrains of poliovirus, B. pertussis protective antigen, diphtheriatoxoid and tetanus toxoid. As mentioned above, the B. pertussisprotective antigen, diphtheria toxoid and tetanus toxoid are availablecommercially as diphtheria-tetanus-pertussis combined vaccines.Therefore, the combined vaccine of the present invention may also beprepared by mixing inactivated Sabin strains of poliovirus together witha diphtheria-tetanus-pertussis combined vaccine.

The inactivated Sabin strains of poliovirus may be prepared by mixinginactivated Sabin type I poliovirus strains, inactivated Sabin type IIpoliovirus strains, and inactivated Sabin type III poliovirus strains.As mentioned above, the inactivated Sabin strains of poliovirus containthe type I, type II and type III inactivated Sabin strains of poliovirusin a ratio, based on the amounts of the respective D antigens thereof,of preferably (2 to 4):(80 to 120):(80 to 120), and most preferably(about 3):(about 100):(about 100).

The B. pertussis protective antigen, diphtheria toxoid and tetanustoxoid may included within the combined vaccine of the invention in anyamounts that are effective for preventing pertussis, diphtheria andtetanus. Specifically, these respective amounts may be the same as thecorresponding amounts in the above-mentioned commercially availablediphtheria-tetanus-pertussis combined vaccines. In cases where theimmunogenicities of the B. pertussis protective antigen, diphtheriatoxoid and/or tetanus toxoid are influenced by the inactivated Sabinstrain of poliovirus and other ingredients, a combined vaccine effectivefor preventing each of the target illnesses may be produced by suitablyadjusting the respective contents.

The combined vaccine of the invention may be produced and used byconventional means. Specifically, production and use may be carried outas described below.

The combined vaccine of the invention may be prepared as an injection bya conventional method. Such an injection is prepared in accordance witha method that is itself known to the art, such as dissolving, suspendingor emulsifying the above substances in a sterile aqueous or oleaginousliquid commonly used in injections. Examples of aqueous liquids forinjection which may be used include physiological saline and isotonicsolutions containing glucose or some other adminiculum. The injectablesolution that has been prepared is typically filled into a suitableampule or syringe.

The combined vaccine of the invention may also optionally includepharmaceutical additives such as preservatives, antioxidants andchelating agents. Illustrative examples of preservatives includethimerosal and 2-phenoxyethanol. Illustrative examples of chelatingagents include ethylenediaminetetraacetic acid and glycol etherdiaminetetraacetic acid.

The combined vaccine of the invention may additionally containadjuvants. Illustrative examples of adjuvants include aluminumhydroxide, aluminum phosphate and aluminum chloride.

In addition to the inactivated Sabin strain poliovirus, B. pertussisprotective antigen, diphtheria toxoid and tetanus toxoid, the combinedvaccine of the invention may also include other immunogenic ingredients.Illustrative examples of such immunogenic ingredients includeimmunogenic ingredients for viruses or bacteria other than poliovirus,B. pertussis, C. diphtheriae and C. tetani. Examples of such immunogenicingredients include toxoids, attenuated viruses, inactivated viruses,proteins, peptides, polysaccharides, lipopolysaccharides, lipopeptidesand combinations thereof. Examples of viruses and bacteria other thanpoliovirus, B. pertussis, C. diphtheriae and C. tetani include influenzaviruses, measles virus, mumps virus, rubella virus, herpes virus,smallpox virus, rabies virus, human immune deficiency virus, hepatitisvirus, Diplococcus pneumoniae, Neisseria meningitidis, typhoid bacillusand Haemophilus influenzae Type b.

The combined vaccine of the present invention may be administeredparenterally, such as by subcutaneous injection or intramuscularinjection, and preferably by subcutaneous injection.

The amount of a single dose of the combined vaccine of the invention maybe suitably selected according to various conditions, such as the ageand body weight of the target vaccinee. Specifically, a single dose maycontain, for example, at least 4 international units of the B. pertussisprotective antigen, about 15 Lf of diphtheria toxoid, about 2.5 Lf oftetanus toxoid, about 2 to about 4 units (preferably about 3 units) ofinactivated Sabin type I poliovirus (D antigen basis), about 80 to about120 units (preferably about 100 units) of inactivated Sabin type IIpoliovirus (D antigen basis), and from about 80 to about 120 units(preferably about 100 units) of inactivated Sabin type III poliovirus (Dantigen basis).

The number of times the combined vaccine of the invention isadministered as an initial immunization may be, for example, two orthree doses at 3- to 8-week intervals. When the inventive combinedvaccine is administered twice as an initial immunization, it isdesirable to administer also a diphtheria-pertussis-tetanus combinedvaccine (DPT vaccine) at an interval of 3 to 8 weeks. As a boosterimmunization, the inventive combined vaccine may be given one more timeat an interval of at least 6 months following initial immunization(e.g., from 12 to 18 months following the end of initial immunization).

EXAMPLES

The present invention is illustrated more fully below by way ofexamples, although the examples do not limit the scope of the invention.

Reference Example 1 Establishing a Manufacturer's Working Cell Bank forPolio Vaccine Viruses

A bank of preserved cells for the production of polio vaccine viruseswas prepared by the procedure described below from Vero cells acquiredfrom ATCC.

(i) Preparation of Master Cell Bank (MCB)

Frozen cells in an ampule received from ATCC (CCL 81 Vero, F-6573;passage number, 124) were thawed, and transferred to an empty 4-ounceflask (a flask having a capacity of 154 mL and a cell growth surfacearea of 54 cm²). Fifteen milliliters of a cell growth medium (DME(Dulbecco's Modified Eagle's Medium; Sigma, catalog No. D5523)containing 5 vol % calf serum, 0.075% sodium bicarbonate, 20 μg/mLerythromycin and 100 μg/mL kanamycin (final concentrations)) was addeddropwise to the cell-containing 4-ounce flask over a period of about 5minutes. The flask containing the cells and the cell growth medium wasstatic cultured at 36° C. (one 4-ounce flask, passage 125). The nextmorning, the cell growth culture was replaced with 15 mL of freshculture and static culturing was again carried out at 36° C. On day 6following the start of the static culture, a subculture was carried out(from one 4-ounce flask to four 4-ounce flasks, passage 126). Thesubculture method was carried out as follows.

Subculture Method

-   (1) Discard culture broth.-   (2) Place 5 mL of 0.25% trypsin solution for subculturing in 4-ounce    flask.-   (3) Immerse cell surfaces for about 1 minute, then discard 0.25%    trypsin solution for subculturing.-   (4) Place 4-ounce flask at rest at 36° C., and wait for cells to    detach from glass surface.-   (5) When cells have begun to detach, add 5 mL of cell growth medium    and induce all cells to detach by pipetting.-   (6) Suspend cells uniformly by further pipetting, then transfer cell    growth medium to a centrifuge tube.-   (7) Centrifuge for 5 minutes at 600 rpm, discard supernatant, and    uniformly suspend sedimented cells in about 8 mL of fresh cell    growth medium by pipetting.-   (8) Add 2 mL of cell suspension to each of four new 4-ounce flasks    (in each of which 13 mL of fresh cell growth medium has been    distributed).-   (9) Place four 4-ounce flasks at rest at 36° C. and carry out cell    cultivation.    The composition of the 0.25% trypsin solution for subculturing was    as follows.

5% trypsin*¹, 50 mL/L

5% Polyvinyl pyrrolidone (90K), 20 mL/L

0.247 mol sodium edetate*², 56 mL/L

EK*³, 2 mL/L

Trypsin diluting fluid*⁴, 872 mL/L

-   *1: Trypsin from porcine pancreas and having an activity of 1:300    was used.-   *2: The composition of 0.247 mol sodium edetate was as follows:

Sodium edetate-2Na.2H₂O, 91.95 g/L

NaOH, 9.88 g/L

-   *3: The composition of EK was as follows:

Erythromycin lactobionate, 10,000 μg/mL

Kanamycin sulfate, 50,000 μg/mL

-   *4: The composition of the trypsin diluting fluid was as follows:

NaCl, 8,000 mg/L

KCl, 400 mg/L

Na₂HPO₄.12H₂O, 150 mg/L

KH₂PO₄, 60 mg/L

Subculturing was subsequently carried out by the same method (although,because the culture surface area is increased about 3- to 4-fold in asingle passage, the culture bottles and the liquid volume handleddiffered) at 3- to 6-day intervals, thereby producing passage 129 cells(the passage number increases by one each time subculturing is carriedout). The cultured passage 129 cells were trypsin treated in 33 SRflasks (Small Roux flasks: culture flasks having a volume of 727 mL anda cell growth surface area of 156 cm²) in the same way as duringsubculturing and centrifugation was carried out, following which thesediment was re-suspended to a concentration of about 1.5×10⁷ cells/mLin a cryopreserved medium (DME (Dulbecco's Modified Eagle's Medium;Sigma, Catalog No. D5523) containing 10% dimethyl sulfoxide (DMSO), 10vol % calf serum, 0.075% sodium bicarbonate, 20 μg/mL erythromycin and100 μg/mL kanamycin (final concentrations)). One milliliter of the abovecell suspension was dispensed to an ampule, the temperature was loweredto −32° C. in a slow freezer (at a cooling rate of about 1° C./min),then transferred to liquid nitrogen and preserved. The passage 129 cellsobtained as described above were used as the master cell bank (MCB).

(ii) Preparation of Manufacturer's Working Cell Bank (MWCB)

Using the Master Cell Bank (MCB) prepared and preserved in section (i)above, the steps from thawing of the cells in the ampule to growth bycell subculturing up to passage 134 were carried out in basically thesame exact way as was used to prepare the Master Cell Bank (MCB) in (i)above (although, because the number of starting cells was higher, theamount of liquid handled and the type and number of culture flasksdiffered). The passage 134 cells were preserved in liquid nitrogen inthe same way as the Master Cell Bank (MCB). The passage 134 cells thusobtained were used as a Manufacturer's Working Cell Bank (MWCB).

Example 1 Preparation of Cells for Production of Polio Vaccine Viruses

(i) Static Cultivation Step

One ampule of the Manufacturer's Working Cell Bank (MWCB) prepared andpreserved in Reference Example 1 above (passage 134 cells) was thawed inthe same way as in the preparation of the Master Cell Bank (MCB) and theManufacturer's Working Cell Bank (MWCB) in Reference Example 1, and thecells were static cultured for 7 days (passage 135) in three LR flasks(Large Roux flasks, which are culture flasks having a capacity of about1,540 mL and a cell growth surface area of about 274 cm²). Subculturingwas carried out until day 7 from the start of static culturing,following which the scale of cultivation was expanded to 18 LR flasks(passage 136) and static culturing was carried out. Static culturing andsubculturing thereof were carried out by the same method as in thepreparation of the Master Cell Bank (MCB) and the preparation of theManufacturer's Working Cell Bank (MWCB) in Reference Example 1 above.

Next, 7 days of static culturing was carried out in a 40-tray CellFactory (Nunc, Catalog No. 139446) (passage 137). Subculturing was thencarried out on day 7 following the start of the static culture, inaddition to which 7 days of static culturing was carried out in four40-tray Cell Factories (passage 138).

(ii) Microcarrier Culturing Step

Next, the passage 138 cells obtained in the static culturing step in (i)above were trypsin treated and centrifuged in the same way as duringsubculturing in (i) above, and the sedimented cells were uniformlysuspended by pipetting in 1,000 mL of a cell growth medium formicrocarrier culturing (DME (Dulbecco's Modified Eagle's Medium; Sigma,Catalog No. D5523) containing 5 vol % calf serum (Thermo Trace), 0.11%sodium bicarbonate, 0.1% fructose, 20 μg/mL erythromycin, and 100 μg/mLkanamycin (final concentrations)). The cell suspension was swelledbeforehand with phosphate buffered saline (PBS), then mixed with amicrocarrier (CYTODEX 1 (trade name); GE Healthcare Biosciences)equilibrated with the cell growth medium for microcarrier culturing (5g/L of CYTODEX 1 (trade name) was used, based on the weight prior toswelling), and culturing was carried out in three 50-liter culturevessels at 37° C., pH 7.15 and under stirring. Starting on day 2 ofculturing, one-half of the cell growth medium was successively replacedonce daily with fresh cell growth medium. The cells grown for 7 dayswere used as cells (passage 139) for the production of polio vaccineviruses.

Example 2 Production of Inactivated Polio Vaccine Type I

(i) Virus Cultivation Step

Just prior to the inoculation of seed viruses into the cells for poliovaccine virus production obtained in Example 1 (passage 139), stirringwas stopped and the cells were allowed to settle, then washed once usingEarl's Balanced Salt Solution (EBSS) containing 0.075% sodiumbicarbonate, 20 μg/mL erythromycin and 100 μg/mL of kanamycin (finalconcentrations). After removing the supernatant from the 5 mL of cellgrowth medium collected together with the microcarrier, the volume wasagain brought up to 5 mL by adding 0.25% trypsin solution, therebydetaching the cells from the beads and causing them to be suspended. Thecell count was then determined, based on which the number of cells forthe entire 50-liter culture vessel was estimated. Attenuated Sabin typeI (LSc, 2ab strains) seed viruses were inoculated in a concentration ofabout 10⁻¹ CCID₅₀ per cell. Following inoculation of the seed viruses,50 L of a virus culture medium (M199 (Medium 199) containing 0.3% sodiumbicarbonate, 20 μg/mL erythromycin and 100 μg/mL of kanamycin (finalconcentrations)) was immediately poured into the culture vessel. Theseed viruses used were viruses that had been cultured beforehand atabout 33.3° C. using African green monkey primary-cultured kidney cells,then packaged in small portions and cryopreserved at −70° C.

Virus cultivation was carried out at 34° C.±1° C. for 3 days. Using thecytopathic effects by the poliovirus (rounding of thepoliovirus-infected cells, followed by detachment of the cells from themicrocarrier) as the indicator, virus cultivation was brought to an endwhen from 95 to 100% of the cells had detached from the microcarrier.Following the completion of virus cultivation, the microcarrier wasremoved with a Teflon mesh (pore size, 120 μm), and the virus suspensionwas recovered. The microcarrier remaining on the mesh was washed oncewith about 3 L of virus culture medium per 50 L culture vessel. Theresulting wash fluid was added to the recovered virus suspension,thereby giving a “Type I polio virus fluid.”

(ii) Virus Concentration/Purification Step

About 150 L of the Type I poliovirus fluid obtained in (i) above waspassed through a 0.2 μm filter membrane (Pall Corporation, SLK7002NRP)to remove cell debris. The filtrate was concentrated to 1.2 L with anultrafiltration membrane (Sartorius, PESU (polyethersulfone) 100 kDa,0.1m², 3051466801E—SG). The concentrated virus fluid was formed into apellet by 4 hours of ultracentrifugation at 6° C. and 100,000 g, andfollowing which the pellet was re-suspended in 0.1 mol/L phosphatebuffer (PB) (the pellet from one centrifuge tube (about 100 mL) wasre-suspended in 5 mL of PB). The pellet re-suspension was shakenovernight at 4° C., then sonicated (Kubota, Insonator Model 200M) for 8minutes at 200 W, thereby breaking up the mass of aggregated viruses.Next, after 30 minutes of centrifugation at 15,000 rpm, the supernatantwas collected. The supernatant thus obtained was purified with DEAESEPHAROSE CL-6B (trade name, GE Healthcare Biosciences; GE 17-0710-05).Phosphate buffer (PB), 0.1 mol/L, was used as the eluate. The absorbanceat 280 nm was monitored and the first peak was recovered as “purifiedType I poliovirus fluid.” The absorbance at 260/280 nm for the sampledpeak was calculated and confirmed to be greater than 1.5 (the 260/280 nmabsorbance for complete poliovirus particles is from 1.6 to 1.7).

(iii) Inactivation Step

The purified type I poliovirus fluid obtained in (ii) above was dilutedabout 10-fold with a pre-inactivation diluting solution (M199 containing5% aminoacetic acid (final concentration), but containing no calcium,magnesium, Phenol Red or sodium bicarbonate), passed through a 0.2 μmfilter membrane (Pall Corporation, SLK7002NRP), and the mass ofaggregated viruses was removed. Following preparation of the filtrate,inactivation was rapidly begun in such a way as to avoid aggregating ofviruses again. One hour prior to the start of inactivation, the filtrateand formalin diluted to 1:200 were separately warmed at 37° C. Whilethoroughly stirring the filtrate, the formalin was added to a finalconcentration of 1:4,000, the mixture was warmed to 37° C., andinactivation was begun. During formalin treatment, virus inactivationwas made to proceed uniformly by stirring the mixture twice daily—oncein the first half of the day and once in the second half of the day.Anticipating that insufficiently inactivated viruses would adhere to thevessel stopper and to certain specific places within the vessel, thestopper was changed on days 2 and 4 following the start of inactivation,and the vessel itself was changed on day 6. In addition, given thepossibility that the viruses would aggregate during the inactivationstep, on day 6 of inactivation, filtration was carried out using a 0.2μm filter membrane (Pall Corporation, SLK7002NRP). The formalintreatment step was brought to an end after 12 days. On day 12, freeformalin in the formalin-treated virus fluid was neutralized with sodiumsulfite (added to a concentration of 0.0264 mol/L), following whichsodium edetate was added (0.0009 mol/L) as a stabilizer, thereby givinga bulk material of “inactivated type I polio vaccine.”

(iv) The amount of D antigen is measured by an indirect ELISA methodusing antibodies having a high specificity to the type and the Dantigen. The indirect ELISA method begins by coating a microplate with,as the primary antibody, a monoclonal antibody (mouse-derived) specificfor D antigens of the same type as the antigen to be assayed. Theantigen to be assayed is then diluted and placed thereon. Next, a rabbitpolyclonal antibody of the same type as the antigen to be assayed isplaced thereon as the secondary antibody, in addition to whichHRPO-labeled anti-rabbit IgG antibody is placed thereon, effecting areaction. Following the reaction, color development is carried out usingan o-phenylenediamine solution, and the absorbance at 492 nm ismeasured. The amount of D antigen (assayed antigen) is determined bycomparing the measured absorbance for the assayed antibody and themeasured absorbance for a reference antigen by parallel-linequantitative analysis.

Example 3 Production of Inactivated Polio Vaccine Type II

Aside from using attenuated Sabin type II strains (P712, Ch, 2abstrains) instead of attenuated Sabin type I strains (LSc, 2ab strains),a bulk material of “inactivated type II polio vaccine” was produced inthe same way as in Example 2.

Example 4 Production of Inactivated Polio Vaccine Type III

Aside from using attenuated Sabin type III strains (Leon, 12a₁b strains)instead of attenuated Sabin type I strains (LSc, 2ab strains), a bulkmaterial of “inactivated type III polio vaccine” was produced in thesame way as in Example 2.

Example 5 Production of B. pertussis Protective Antigen

(1) Cultivation of Bordetella Pertussis

Strains of phase I B. pertussis (Tohama strain) were spin-cultured at 30to 34° C. for 20 to 24 hours in a Cohen-Wheeler medium. The B. pertussisgrown on the Cohen-Wheeler medium were then grown on a Stainer-Scholtemedium at 30 to 34° C. for 48 to 68 hours.

The culture was concentrated to 1/10^(th) the original volume byultracentrifugation, and the supernatant and bacterial cells wereseparated by centrifugal separation.

(2) Preparation of Pertussis Toxin

Next, 1 mol/L phosphate buffer (pH 8.0) was added to the supernatantobtained in (1) above so as to bring the volume up to 1/10^(th) theoriginal volume, then a 25 w/v % calcium acetate solution was added tobring the concentration to from 0.1 to 2.0 w/v %, and a filtratecontaining pertussis toxin (phylactic antigen) was obtained byfiltration. The filtrate was passed through a column by SP columnchromatography (equilibrated solution: 0.1 mol/L phosphate buffer (pH6.0) and the adsorbed pertussis toxin was eluted with 0.415 mol/Lphosphate buffer (pH 7.0), thereby fractionating a pertussistoxin-containing solution. Next, the pertussis toxin-containing solutionwas passed through a column by gel column chromatography (equilibratedsolution: 0.025 mol/L sodium phosphate solution (pH 8.7) containing 0.25mol/L sodium chloride), then filtered (pore size, 0.2 μm), giving a purepertussis toxin (PT antigen) solution.

(3) Preparation of Filamentous Hemagglutinin

The bacterial cells isolated in (1) above were dispersed in a 0.05 mol/Lphosphate buffer (pH 8.0) containing 1 mol/L sodium chloride, followingwhich centrifugal separation was carried out to again separate thesupernatant and the bacterial cells. A 25 w/v % calcium acetate solutionwas added to the re-separated supernatant to a concentration of from 0.1to 2.0 w/v %, following which filtration was carried out, giving asolution containing filamentous hemagglutinin (phylactic antigen). Thefiltrate was concentrated with ammonium sulfate, then purified bydensity gradient centrifugation, thereby fractionating pure filamentoushemagglutinin (FHA antigen).

(4) Preparation of Outer Membrane Protein

The bacterial cells re-separated in (3) above were dispersed in a 0.01mol/L phosphate buffer (pH 7.0) containing 0.145 mol/L of sodiumchloride and heated at 60° C. for 90 minutes, following which thesupernatant and bacterial cells were again re-separated by centrifugalseparation. Next, 1 mol/L phosphate buffer (pH 8.0) was added to theagain re-separated supernatant so as to bring the volume up to1/10^(th), following which a 25 w/v % calcium acetate solution was addedto concentrations of from 0.1 to 2.0 w/v % and filtration was carriedout, thereby giving a filtrate containing outer membrane protein(phylactic antigen). The solution containing outer membrane protein(phylactic antigen) was subjected to SP column chromatography(equilibrated solution: 0.1 mol/L phosphate buffer (pH 6.0)), and theliquid that passed through the column was collected. Next, the liquidwas passed through a column by gel column chromatography (equilibratedsolution: 0.025 mol/L sodium phosphate solution (pH 8.7) containing 0.25mol/L sodium phosphate), then filtered (pore size, 0.2 μm), therebygiving pure outer membrane protein (69 K antigen).

(5) Preparation of Fimbriae (Agglutinogen (AGG))

A 0.1 mol/L phosphate buffer (pH 8.0) containing 1 mol/L of sodiumchloride was added to the residue left following collection of the outermembrane protein-containing solution in (4) above in an amount 1/10^(th)the amount of the supernatant, following which filtration was carriedout, yielding a filtrate containing fimbriae (phylactic antigen). Theresulting fimbriae-containing solution was concentrated with ammoniumsulfate, then purified by density gradient centrifugation, therebyfractionating pure fimbriae (FB antigen).

(6) Preparation of Protective Antigen (Attenuation)

A solution containing the PT antigen, FHA antigen, 69 KD antigen and FBantigen obtained in sections (2) to (5) above mixed in such a way thatthe DPT combined vaccine prepared in Example 8 below will includeantigen levels of 1.89 μg of PT antigen, 3.00 μg of FHA antigen, 0.76 μgof 69K antigen and 0.36 μg of FB antigen per 0.5 mL of vaccine wasprepared as a pure phylactic antigen solution. Formalin (from 0.2 to 0.5vol %) and, if necessary, lysine hydrochloride to a concentration of notmore than 1 w/v % were added to the pure phylactic antigen solution,following which the solution was warmed at 37 to 41° C. for at least 7days to effect attenuation, thereby giving a pertussis protectiveantigen solution. Excess formalin and lysine hydrochloride were removedby ultrafiltration, giving a pertussis protective antigen bulk material.

Example 6 Production of Diphtheria Toxoid

(1) Cultivation of Corynebacterium Diphtheriae

C. diphtheriae (Park-Williams No. 8 strain) was cultured on Loeffler'smedium at 32.0 to 34.0° C. for 5 days.

(2) Preparation of Diphtheria Toxin

Ammonium sulfate was added to the culture fluid obtained in (1) above,following which the supernatant was filtered (pore size, 0.45 μm). Theresulting filtrate was passed through a column by phenyl hydrophobiccolumn chromatography (equilibrated solution: 0.01 mol/L sodiumphosphate solution (pH 6.5) containing 1.25 mol/L ammonium sulfate). Thetoxin solution thereby obtained was passed through a column by DEAEion-exchange column chromatography (equilibrated solution: 0.01 mol/Lphosphate buffer (pH 7.0)), then passed through a column by gel columnchromatography (equilibrated solution: 0.1 mol/L phosphate buffer (pH7.0) containing 0.145 mol/L sodium chloride), thereby fractionating thediphtheria toxin. This was used as a pure toxin solution.

(3) Conversion of Diphtheria Toxin to Toxoid

Next, lysine hydrochloride was added to the pure toxin solution obtainedin above (2) to a concentration of not more than 1 w/v %, formalin wasadded to a concentration of 0.3 vol %, and the solution was warmed at38.0 to 40.0° C. for 21 days, thereby converting the toxin to toxoid.

(4) Preparation of Diphtheria Toxoid

Following the conversion to toxoid in (3) above, excess formalin andlysine hydrochloride were removed by ultrafiltration, thereby giving adiphtheria toxoid.

Example 7 Production of Tetanus Toxoid

(1) Cultivation of Clostridium Tetani

C. tetani (Harvard 47-A) was cultured in liver bouillon at 34.5° C. to36.5° C. for 5 days.

(2) Preparation of Tetanus Toxin

Ammonium sulfate was added to the culture obtained in (1) above to aconcentration of 1.25 mol/L per liter of the culture. Next, the culturewas passed through a column by phenyl hydrophobic column chromatography(equilibrated solution: 0.01 mol/L sodium phosphate (pH 6.5) containing1.25 mol/L ammonium sulfate). The resulting toxin solution was passedthrough a column by DEAE ion-exchange chromatography (equilibratedsolution: 0.01 mol/L phosphate buffer (pH 7.5)), following which thesolution was passed through a column by gel column chromatography(equilibrated solution: 0.004 mol/L phosphate buffer (pH 7.0) containing0.145 mol/L sodium chloride), thereby fractionating the tetanus toxin.This was used as a pure toxin solution.

(3) Conversion of Tetanus Toxin to Toxoid

Next, formalin was added to the pure toxin solution obtained in above(2) to a concentration of 0.3 vol %, the pH was corrected to 7.0, andthe solution was warmed at 39° C. for 15 to 23 days, thereby convertingthe toxin to toxoid.

(4) Preparation of Tetanus Toxoid

Following the conversion to toxoid in (3) above, excess formalin andlysine hydrochloride were removed by ultrafiltration, thereby giving atetanus toxoid.

Example 8 Production of Combined Vaccine

(i) Preparation of Combined Inactivated Polio Vaccine

The bulk materials of inactivated polio vaccine type I, inactivatedpolio vaccine type II and inactivated polio vaccine type III obtained inExamples 2, 3 and 4 above were mixed with medium 199 (M199),2-phenoxyethanol (final concentration, 0.5 vol %) and aluminum chloride(final concentration, 0.09 w/v %) in such a way as to set the relativelevels of the respective D antigens to 3:100:100. The resulting mixturewas adjusted to a pH of 7 with sodium hydroxide or hydrochloric acid,thereby giving a combined inactivated polio vaccine.

(ii) Production of DPT Combined Vaccine

The B. pertussis protective antigen, diphtheria toxoid and tetanustoxoid bulk materials obtained in Examples 5, 6 and 7 above were mixedwith 199 medium (M199), 2-phenoxyethanol (final concentration, 0.5 vol%) and aluminum chloride (final concentration, 0.24 w/v %). Theresulting mixture was adjusted to a pH of 7 with sodium hydroxide andhydrochloric acid, thereby giving a DPT combined vaccine.

(iii) Production of Combined Vaccine

The combined inactivated polio vaccine obtained in (i) above and the DPTcombined vaccine obtained in (ii) above were mixed in equal amounts,thereby producing a combined vaccine.

Example 9 Stability of Combined Vaccine

The combined vaccine obtained in Example 8 was measured by carrying outan accelerated test at 25° C. The stability was evaluated by potencytests on each of the viruses.

(1) Potency Test of Precipitated Pure Pertussis Vaccine

A test specimen, a standard pertussis vaccine and B. pertussis strain18323 are used. The test specimen and the standard vaccine are eachdiluted, from which dilutions of each in a total of at least 3 serialdilutions at suitable logarithmically equal intervals of 4-fold or moreare then prepared. One group of at least sixteen 4-week-old mice is usedfor each dilution. Each animal is given 0.5 mL of dilution as a singleintraperitoneal injection. Twenty-one days after the immune injections,0.025 mL of a bacterial cell suspension for challenge is injected intothe brain of each animal, following which the animals are observed for14 days and the number of deaths is tallied. From statistical treatmentand comparison of the test results, the test specimen must have apotency of at least 8 units/mL.

(2) Potency Test of Precipitated Diphtheria Toxoid

A test specimen, a control precipitated diphtheria toxoid and a suitabletoxin solution are used. Dilution of the test specimen and the controlis carried out with physiological saline; dilution of the toxin solutionis carried out with a 0.017 mol/L phosphate buffer/sodium chloridesolution (pH 7.0) containing 0.2 w/v % of gelatin. The test specimen andthe control are each diluted, creating serial dilutions atlogarithmically equal intervals. Using one group of at least ten5-week-old mice for each dilution of the test specimen and the control,each animals is subcutaneously injected with 0.5 mL. Four to six weeksfollowing the immune injections, blood is drawn from each animal, andthe blood antitoxin titer is measured. From statistical treatment andcomparison of the test results, the test specimen must have a potency ofat least 47 units/mL.

(3) Potency Test of Precipitated Tetanus Toxoid

A test specimen, a control precipitated tetanus toxoid and a suitabletoxin solution are used. Dilution of the test specimen and the controlis carried out with physiological saline; dilution of the toxin solutionis carried out with a 0.017 mol/L phosphate buffer/sodium chloridesolution (pH 7.0) containing 0.2 w/v % of gelatin. The test specimen andthe control are each diluted, creating serial dilutions atlogarithmically equal intervals. Using one group of at least ten5-week-old mice for each dilution of the test specimen and the control,each animal is given a single subcutaneous injection of 0.5 mL. Four tosix weeks following the immune injections, each mouse is challenged withabout 100 LD₅₀ of toxin and observed for 4 days. From statisticaltreatment and comparison of the test results, the test specimen musthave a potency of at least 27 units/mL.

(4) Rat Immunogenicity Test

A test specimen, a control for an IPV potency test, standard sera foreach type, and Sabin strains of poliovirus (types I, II and III) forneutralization test challenges are used. The test specimen and controlare each diluted, creating dilutions at logarithmically equal intervals.One group of at least ten Wister 8-week-old female rats is used for eachdilution. Each animal is intramuscularly inoculated with 0.5 mL in thefemoral region of the hind leg. Twenty-one days following inoculation,blood is drawn individually from each animal and the serum is collectedthen heated at 56° C. for 30 minutes. Serum for each animal and thestandard serum are placed in at least two wells for each serum, and2-fold serially diluted with MEM medium. In addition, the respectivewells are inoculated to about 100 CCID₅₀ with neutralizing virussuspensions of the respective types. Next, all the plates are placed ina 36±1° C. CO₂ incubator for 3 hours, then allowed to react overnight atabout 4° C. Cell suspension containing 1×10⁴ cells is added thefollowing day to each well, and is cultured in a 36±1° C. CO₂ incubatorfor 7 days. After the completion of culturing, the CPE for each well areexamined, the serum dilution ratio at the time of 50% neutralization iscalculated, and the reciprocal thereof is treated as the neutralizingantibody titer. From statistical treatment and comparison of the testresults, the test specimen must have a potency equal to or higher thanthe control.

Results for the accelerated test are shown in Table 1. Lot 04C (JapanPolio Research Institute) was used as the control IPV.

TABLE 1 At start 1 2 3 Specification of test month months monthsInactivated polio vaccine Equal to or higher than 1.7 1.7 1.5 2.1 type Icontrol IPV Inactivated polio vaccine Equal to or higher than 2.1 2.01.6 1.5 type II control IPV Inactivated polio vaccine Equal to or higherthan 20.3 12.3 5.9 6.0 type III control IPV Precipitated pure pertussis 8 units/mL 15.8 13.8 14.6 27.3 vaccine Precipitated diphtheria 47units/mL 97.9 109.3 85.9 102.9 toxoid Precipitated tetanus toxoid 27units/mL 72.3 88.6 105.5 79.4

Example 10 Vero Cell Cultivation by Microcarrier Method, and PoliovirusCultivation

(i) Vero Cell Cultivation

Cells that had been subcultured in a static culture starting from aworking bank of Vero cells (MWCB93) were detached with a trypsin-EDTAsolution (0.25% trypsin, 0.14 M EDTA), then centrifuged at 600 rpm for10 minutes and subsequently suspended in a cell growth medium(Dulbecco's modified Eagle medium (DME) containing 5 vol % calf serum,0.11% sodium bicarbonate (NaHCO₃), 0.1% fructose, 20 μg/mL erythromycin,and 100 μg/mL kanamycin).

A microcarrier (CYTODEX 1 (trade name) was swelled in PBS (−), autoclavesterilized at 121° C. for 15 minutes, then substituted with cell growthmedium and used.

CELLIGEN Plus and CELLIGEN bioreactors manufactured by New BrunswickScientific were used as the culture apparatuses. The microcarrier andcell suspension were added to the bioreactor, following which the cellgrowth medium was added to a final volume of 4.8 L (in the case of theCELLIGEN bioreactor, 3.5 L) and culturing was carried out at atemperature of 37.0° C., a dissolved oxygen concentration (DO) of 15%, apH of 7.15 and a rotational speed of 35 to 50 rpm. Medium exchange usingcell growth medium having a NaHCO₃ at the concentration of 0.15% as theexchange medium was carried out continuously from day 2 of culturing ata rate of about 4 L/day (in the case of the Celligen bioreactor, theentire amount of medium was exchanged once every other day). The cellcount was measured using a COULTER COUNTER (trade name).

The cell culturing results are shown in FIG. 1 (Celligen bioreactorswere used only in the 3H tests). At a carrier concentration of 3 g/L,the cell count reached a maximum on day 7 at a low-concentrationstarting cell number (3 L, about 2×10⁵ cells/mL) and reached a maximumon day 8 at a high-concentration starting cell number (3H, about 10×10⁵cells/mL), after which it decreased. At a carrier concentration of 5g/L, the cell count increased over a period of 11 days at alow-concentration starting cell number (5 L, about 2×10⁵ cells/mL), butthe cell count decreased on day 12. In the case of a low-concentrationstarting cell number, the total cell count at a carrier concentration of5 g/L was greater on day 10 than the total cell count at a carrier levelof 3 g/L, but the difference was not large. At a carrier concentrationof 5 g/L and a high-concentration starting cell number (5H, about 10×10⁵cells/mL), the cell count was still increasing on day 9, with the cellcount at that point being about 2.8×10⁶ cells/mL, representing anincrease of about 2.7 times over the starting cell number.

(ii) Culturing of Poliovirus

A type I poliovirus (IS-90C) was used as the seed virus. On the finalday of cell count measurements, the cells were washed with a 4-foldvolume (based on culturing capacity of bioreactor) of EBSS containing0.075% NaHCO₃. The seed virus was then diluted in 1 liter of M-199 (E)medium containing 0.3% NaHCO₃, 20 μg/mL erythromycin and 100 μg/mLkanamycin (virus growth medium). The resulting virus dilution was theninoculated into the washed cells, and the virus culture medium was addedup to the culturing capacities of the respective bioreactors. Viruscultivation was carried out at a culturing temperature of 33.3° C., 15%dissolved oxygen (DO), a pH of 7.40 and a rotational speed of 35 to 50rpm. Virus cultivation was stopped when the cells had completelydetached from the microcarrier due to the cytopathic effects (CPE) ofthe virus, at which point the virus fluid was harvested. The harvestedvirus fluid was cryopreserved at −80° C.

In this test, virus cultivation was begun on the final day ofmeasurement on the respective cell growth curves shown in FIG. 1.

Measurement of the virus titer was carried out as follows. GMK-2 cellscultured for 3 days in a roller tube were washed twice with 1 mL of HBSScontaining 0.075% NaHCO₃, 200 u/mL penicillin and 200 μg/mLstreptomycin, following which 1 mL of a cell maintenance fluid (M-199medium containing 0.1% bovine serum albumin, 0.225% NaHCO₃, 200 u/mLpenicillin and 200 μg/mL streptomycin) was added. The test virus fluidwas serially diluted 0.5 log₁₀ with the cell maintenance fluid, and 0.2mL of 10⁻⁷ to 10^(−8.5) virus fluid per tube was inoculated in 5 tubesat each dilution level. Following inoculation, culturing was carried outfor 7 days in a 36° C. incubator. The infectivity titer (CCID₅₀/0.2 mL)was computed by the Reed & Muench method based on a judgment fromobservation of the cytopathic effects (CPE) on day 7.

FIG. 2 shows the infectivity titers (virus titers) of type I poliovirusobtained in the Vero cells cultured under various conditions. As aresult of virus cultivation, the highest infectivity titer was obtainedin the system having a carrier concentration of 5 g/L and ahigh-concentration starting cell number (5H); this system reached a celldensity on day 9 of 2.8×10⁶ cells/mL. The systems, arranged according tothe size of the infectivity titers thereof in descending order, were 5H,5L and 3L. These results were in agreement with the total cell counts.In addition, compared with the virus fluid obtained by cultivation ingreen monkey kidney cells as the control, an infectivity titer more than10 times higher was obtained in the 5H system. The virus titers shown inFIG. 2 (log₁₀ CCID₅₀/0.2 mL) were 8.18 in the 3L system, 8.51 in the 5Lsystem, 8.59 in the 5H system, and 7.50 in Ref (control).

INDUSTRIAL APPLICABILITY

High-potency Sabin strains of polioviruses can be obtained by culturing,in the presence of from about 4 g/L to about 6 g/L of a microcarrier,Vero cells inoculated with Sabin strain polioviruses. Inactivated Sabinstrain polioviruses can be efficiently produced by using high-potencySabin strain polioviruses. Therefore, a process for producing a combinedvaccine which includes the step of culturing, in the presence of fromabout 4 g/L to about 6 g/L of microcarrier, Vero cells inoculated withSabin strain polioviruses (the production process of the presentinvention) is useful as a process for efficiently producing combinedvaccines containing inactivated Sabin strain polioviruses. In addition,because the combined vaccine of the present invention is capable ofeffectively suppressing the onset of polio, pertussis, diphtheria andtetanus, it is useful as a vaccine for polio, pertussis, diphtheria andtetanus.

The invention claimed is:
 1. A process for producing a combined vaccinecontaining, (A) type I, type II, and type III inactivated Sabin strainsof poliovirus in a weight proportion of D antigens of(2-4):(80-120):(80-120), (B) a Bordetella pertussis protective antigen,(C) a diphtheria toxoid, and (D) a tetanus toxoid, the processcomprising: (a) culturing Vero cells in about 4 g/L to about 6 g/L of amicrocarrier, wherein the culture medium is selected from the groupconsisting of ME medium, DME medium, RPMI 1640 medium and RPMI 199medium, and wherein the culture medium contains from about 5 to about 70vol % of calf serum or fetal bovine serum; (b) infecting the Vero cellswith type I, type II and type III Sabin strains of poliovirus; (c)allowing the Sabin strains to proliferate; (d) recovering a virus fluidcontaining the Sabin strains; (e) inactivating the Sabin strains toproduce the type I, type II, and type III inactivated Sabin strains ofpoliovirus of (A); and (f) mixing the inactivated Sabin strains ofpoliovirus of (A with a diphtheria-tetanus-pertussis combined vaccinecomprising the Bordetella pertussis protective antigen, diphtheriatoxoid, and tetanus toxoid of (B)-(D).
 2. The process according to claim1, wherein the microcarrier has a concentration of about 5 g/L.
 3. Theprocess according to claim 1, wherein the microcarrier is a dextranmicrocarrier.
 4. The process according to claim 1, wherein the step ofculturing the Vero cell (step (a)) is carried out on a scale of at leastabout 3 liters.
 5. The process according to claim 1, wherein the step ofculturing the Vero cell (step (a)) is carried out on a scale of at leastabout 30 liters.
 6. The process according to claim 1, further comprising(d-2) a step of purifying the virus fluid.
 7. The process according toclaim 6, wherein the purifying step (step (d-2)) comprises: (i) formingthe virus fluid recovered in step (d) into a pellet byultracentrifugation, (ii) sonicating a re-suspension of the pellet; and(iii) purifying by column chromatography.
 8. The process according toclaim 7, wherein the purification by column chromatography (iii) iscarried out one time only.
 9. A vaccine produced by the processaccording to claim
 1. 10. The process according to claim 1, wherein themicrocarrier has a concentration of about 4.5 g/L to about 5.5 g/L.